Title Page
Contents
ABSTRACT 13
INTRODUCTION 16
1. Recombinant protein production 16
2. Escherichia coli as a cell factory 17
3. Stressors in mass cultivation 18
4. Molecular chaperone 19
5. Heat shock proteins of plants 23
6. Alcohol dehydrogenase 24
7. Purpose of this study 25
MATERIALS AND METHODS 27
Ⅰ. Cell lines and culture condition 27
1. Escherichia coli cell lines 27
2. Culture condition 31
Ⅱ. General procedures 32
1. Protein extraction 32
2. Protein purification 32
3. SDS-PAGE 33
4. Protein quantitative analysis 34
5. rADH activity assay 34
Ⅲ. in vivo studies 35
1. Confirmation of heterologous Hsp expression 35
2. Cell growth and protein quantity and solubility assessment under stress conditions 36
Ⅳ. in vitro studies 38
1. Preparation of purified heterologous Hsps and rADH 38
2. Recombinant ADH enzyme activity assessment 38
3. Recombinant ADH aggregation assay 41
RESULTS AND DISCUSSION 42
Ⅰ. Comparison of enhanced bacterial cell growth and protein accumulation by heterologous expression of Hsps 42
1. Confirmation of the heterologous expression of Hsps 42
2. Enhanced growth rate, protein quantity, and protein solubility by heterologous expression of Hsps 44
Ⅱ. Increased yield, activity, and solubility of recombinant ADH with the interaction with heterologous Hsps 59
1. Increased yield and activity of rADH by co-expression of DcHsp70 59
2. Activity of rADH under thermal stress 62
3. Increased rADH activity by interacting with heterologous Hsps 64
4. Effects of ATP on the activity-enhancing effect of DcHsp70 on rADH under heat conditions 74
5. Increased rADH activity by interacting with double Hsps 76
6. Enhanced rADH solubility by interacting with DcHsp70 under heat conditions 88
CONCLUSION 92
REFERENCES 94
국문 요약 104
Table 1. The list of the genes and plasmid vectors inserted into E. coli BL21(DE3) 30
Table 2. Stress conditions of in vivo studies 36
Figure 1. Graphical depiction of the function of Hsp70 and its cofactors 21
Figure 2. Graphical depiction of the function of sHsp 22
Figure 3. Graphical description of ADH function on ethanol. ADH oxidizes ethanol to acetaldehyde. In this process, NAD⁺ is reduced to NADH, and the... 25
Figure 4. Graphical summary of the Escherichia coli cell lines carrying recombinant plasmid vectors with Hsp genes form carrot and E. coli. The origin of the genes are color-coded with different colors; orange : carrot, black : E. coli.... 28
Figure 5. Graphical summary of the Escherichia coli cell lines expressing ADH with DcHsp70 in the genome. a the site of the genome, yddE pseudogene site,... 29
Figure 6. SDS-PAGE results of purified Hsps. The proteins were expressed by pET vector system in E. coli BL21(DE3) with the induction of 0.5 mM IPTG at... 43
Figure 7. Growth rate and protein analysis results of cell lines expressing Hsps under the optimal condition (37℃). a growth rate, b protein... 46
Figure 8. Growth rate and protein analysis results of cell lines expressing Hsps under the heat condition (47℃). a growth rate, b protein... 49
Figure 9. Growth rate and protein analysis results of cell lines expressing Hsps under the cold condition (16℃). a growth rate, b protein... 52
Figure 10. Growth rate and protein analysis results of cell lines expressing Hsps under the acetate condition (100 mM sodium acetate). a growth rate,... 54
Figure 11. Growth rate and protein analysis results of cell lines expressing Hsps under the ethanol condition (1%). a growth rate, b protein... 56
Figure 12. SDS-PAGE result of purified rADH from the cell lines with or without the co-expression of DcHsp70. The rADH was induced with 0.5 mM of... 60
Figure 13. Comparison of the activity of rADH expressed with or without heterologous expression of DcHsp70. rADH in pET11a plasmid vector in E. coli... 61
Figure 14. rADH activity under heat stress. 50 ㎍ of rADH was exposed to heat stress indicated on the x-axis of the graph for the indicated period. The final... 63
Figure 15. Activity of rADH treated with Hsps at 37℃. 120 nM of rADH and 600, 900, and 1200 nM of Hsps were treated at 37℃ for 30 min. Then, O.D.... 65
Figure 16. Activity of rADH treated with Hsps at 50℃. 120 nM of rADH and 600, 900, and 1200 nM of Hsps were treated at 37℃ for 30 min. Then, O.D.... 67
Figure 17. Activity of rADH treated with Hsps at 55℃. 120 nM of rADH and 600, 900, and 1200 nM of Hsps were treated at 37℃ for 30 min. Then, O.D.... 69
Figure 18. Activity of rADH treated with Hsps at 60℃. 120 nM of rADH and 600, 900, and 1200 nM of Hsps were treated at 37℃ for 30 min. Then, O.D.... 71
Figure 19. Activity of rADH with DcHsp70 with ATP. 120 nM of rADH, 1200 nM of DcHsp70, and 3 mM of ATP were treated at a 37℃, b 45℃, c 50℃ and d... 75
Figure 20. Activity of rADH interacting with heterologous Hsps at 37℃. 120 nM of rADH, 600 nM of sHsps, and 100, 200, and 300 nM of Hsp70s were treated... 78
Figure 21. Activity of rADH interacting with heterologous Hsps at 50℃. 120 nM of rADH, 600 nM of sHsps, and 100, 200, and 300 nM of Hsp70s were treated... 81
Figure 22. Activity of rADH interacting with heterologous Hsps at 55℃. 120 nM of rADH, 600 nM of sHsps, and 100, 200, and 300 nM of Hsp70s were treated... 83
Figure 23. Comparison between single and double treatment of DcHsp17.7 with Hsp70s. a 37℃, b 50℃, c 55℃. Data from Fig. 20, 22, and 23 have been... 85
Figure 24. Comparison between single and double treatment of IbpA with or without Hsp70s. a 37℃, b 50℃, c 55℃. Data from Fig. 20, 22, and 23 have... 86
Figure 25. Comparison between single and double treatment of IbpB with or without Hsp70s. a 37℃, b 50℃, c 55℃. Data from Fig. 20, 22, and 23 have... 87
Figure 26. Aggregation of rADH treated with DcHsp70 under 50℃. 120 and 240 nM of rADH were treated with 600, 900 (only with 120 nM of ADH), and 1200... 91